RESUMEN
BACKGROUND: Coronary artery bypass surgery provides excellent patency rates; however, the early and/or late graft failure reduces the long-term benefit of myocardial revascularization. We investigated the effectiveness of generally used saline, Custodiol solutions and a new solution (TiProtec) at preserving endothelium after cold ischemia and warm reperfusion injury. MATERIALS AND METHODS: Aortic transplantations were performed in Lewis rats. Aortic arches were stored in saline, Custodiol, and TiProtec solutions for 2 h then were transplanted into the abdominal aorta. Two, 24 hours and 1 week after transplantation, the implanted grafts were harvested. Endothelium-dependent and -independent vasorelaxations were investigated in organ bath. DNA strand breaks were assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-method, messenger RNA expressions by quantitative real-time polymerase chain reaction, and the expression of CD-31 and alpha smooth muscle actin by immunochemistry. RESULTS: Severely impaired endothelial function and integrity of implanted aortic grafts were shown after 2 h in the saline, Custodiol group (maximal vasorelaxation to acetylcholine: control: 91 ± 2%, saline: 26 ± 5%, Custodiol: 24 ± 5%, CD-31-positive area control: 96 ± 2%, saline: 35 ± 13% Custodiol: 54 ± 5%, P < 0.05, respectively); however, a preserved endothelial function was observed in the TiProtec group when compared with the saline and Custodiol group (maximal vasorelaxation: 46 ± 7%, CD-31-positive area: 54 ± 10%, P < 0.05). After 1 wk, endothelial function was partially recovered in all groups; however, it was significantly better in the TiProtec group (maximal vasorelaxation to acetylcholine: saline: 42 ± 3%, Custodiol: 48 ± 3%, TiProtec: 56 ± 3%, CD-31-positive area: saline: 56 ± 5%, Custodiol: 54 ± 4%; TiProtec: 83 ± 6%, P < 0.05, respectively). In addition, messenger RNA levels of Bax, B-cell lymphoma-2, endothelial NOS, vascular endothelial growth factor 2, and caspase-3 were significantly altered in both groups. CONCLUSIONS: TiProtec appears to be superior for the preservation of endothelial- and smooth muscle cells of bypass graft after cold storage and warm reperfusion in our murine model.
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Aorta Torácica/trasplante , Endotelio Vascular/efectos de los fármacos , Soluciones Preservantes de Órganos/farmacología , Vasodilatación/efectos de los fármacos , Animales , Aorta Abdominal/cirugía , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiopatología , Isquemia Fría , Puente de Arteria Coronaria , Endotelio Vascular/fisiopatología , Glucosa/farmacología , Etiquetado Corte-Fin in Situ , Masculino , Manitol/farmacología , Cloruro de Potasio/farmacología , Procaína/farmacología , Ratas , Ratas Endogámicas Lew , Reacción en Cadena en Tiempo Real de la Polimerasa , Reperfusión , Cloruro de Sodio/farmacologíaRESUMEN
Storage protocols of vascular grafts need further improvement against ischemia-reperfusion (IR) injury. Hypoxia elicits a variety of complex cellular responses by altering the activity of many signaling pathways, such as the oxygen-dependent prolyl-hyroxylase domain-containing enzyme (PHD). Reduction of PHD activity during hypoxia leads to stabilization and accumulation of hypoxia inducible factor (HIF) 1α. We examined the effects of PHD inhibiton by dimethyloxalylglycine on the vasomotor responses of isolated rat aorta and aortic vascular smooth muscle cells (VSMCs) in a model of cold ischemia/warm reperfusion. Aortic segments underwent 24 hours of cold ischemic preservation in saline or DMOG (dimethyloxalylglycine)-supplemented saline solution. We investigated endothelium-dependent and -independent vasorelaxations. To simulate IR injury, hypochlorite (NaOCl) was added during warm reperfusion. VSMCs were incubated in NaCl or DMOG solution at 4°C for 24 hours after the medium was changed for a supplied standard medium at 37°C for 6 hours. Apoptosis was assessed using the TUNEL method. Gene expression analysis was performed using quantitative real-time polymerase chain reaction. Cold ischemic preservation and NaOCl induced severe endothelial dysfunction, which was significantly improved by DMOG supplementation (maximal relaxation of aortic segments to acetylcholine: control 95% ± 1% versus NaOCl 44% ± 4% versus DMOG 68% ± 5%). Number of TUNEL-positive cell nuclei was significantly higher in the NaOCl group, and DMOG treatment significantly decreased apoptosis. Inducible heme-oxygenase 1 mRNA expressions were significantly higher in the DMOG group. Pharmacological modulation of oxygen sensing system by DMOG in an in vitro model of vascular IR effectively preserved endothelial function. Inhibition of PHDs could therefore be a new therapeutic avenue for protecting endothelium and vascular muscle cells against IR injury.
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Aorta/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Daño por Reperfusión/prevención & control , Vasodilatación/efectos de los fármacos , Aminoácidos Dicarboxílicos/farmacología , Animales , Aorta/enzimología , Aorta/patología , Apoptosis/efectos de los fármacos , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Inhibidores Enzimáticos/farmacología , Hemo-Oxigenasa 1/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Etiquetado Corte-Fin in Situ , Contracción Isométrica/efectos de los fármacos , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/enzimología , Daño por Reperfusión/patologíaRESUMEN
BACKGROUND: Heart valve tissue engineering represents a concept for improving the current methods of valvular heart disease therapy. The aim of this study was to develop tissue engineered heart valves combining human umbilical vein endothelial cells (HUVECs) and decellularized human heart valve matrices. METHODS AND RESULTS: Pulmonary (n=9) and aortic (n=6) human allografts were harvested from explanted hearts from heart transplant recipients and were decellularized using a detergent-based cell extraction method. Analysis of decellularization success was performed with light microscopy, transmission electron microscopy and quantitative analysis of collagen and elastin content. The decellularization method resulted in full removal of native cells while the mechanical stability and the quantitative composition of the neoscaffolds was maintained. The luminal surface of the human matrix could be successfully recellularized with in vitro expanded HUVECs under dynamic flow conditions. The surface appeared as a confluent cell monolayer of positively labeled cells for von Willebrand factor and CD 31, indicating their endothelial nature. CONCLUSIONS: Human heart valves can be decellularized by the described method. Recellularization of the human matrix resulted in the formation of a confluent HUVEC monolayer. The in vitro construction of tissue-engineered heart valves based on decellularized human matrices followed by endothelialization using HUVECs is a feasible and safe method, leading to the development of future clinical strategies in the treatment of heart valve disease.
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Bioprótesis , Prótesis Valvulares Cardíacas , Células Endoteliales de la Vena Umbilical Humana/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , MasculinoRESUMEN
BACKGROUND: Reperfusion of ischemic myocardium may contribute to substantial cardiac tissue damage, but the addition of iron chelators, zinc or zinc complexes has been shown to prevent heart from reperfusion injury. We investigated the possible beneficial effects of an iron-chelating and zinc-complexing agent, Q50, in rat models of ischemia/reperfusion (I/R)-induced myocardial infarction and on global reversible myocardial I/R injury after heart transplantation. METHODS AND RESULTS: Rats underwent 45-min myocardial ischemia by left anterior descending coronary artery ligation followed by 24h reperfusion. Vehicle or Q50 (10 mg/kg, IV) were given 5 min before reperfusion. In a heart transplantation model, donor rats received vehicle or Q50 (30 mg/kg, IV) 1h before the onset of ischemia. In myocardial infarcted rats, increased left ventricular end-systolic and end-diastolic volumes were significantly decreased by Q50 post treatment as compared with the sham group. Moreover, in I/R rat hearts, the decreased dP/dtmax and load-independent contractility parameters were significantly increased after Q50. However, Q50 treatment did not reduce infarct size or have any effect on increased plasma cardiac troponin-T-levels. In the rat model of heart transplantation, 1h after reperfusion, decreased left ventricular systolic pressure, dP/dt(max), dP/dt(min) and myocardial ATP content were significantly increased and myocardial protein expression of superoxide dismutase-1 was upregulated after Q50 treatment. CONCLUSIONS: In 2 experimental models of I/R, administration of Q50 improved myocardial function. Its mechanisms of action implicate in part the restoration of myocardial high-energy phosphates and upregulation of antioxidant enzymes.
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Quelantes del Hierro/farmacología , Daño por Reperfusión Miocárdica , Miocardio/metabolismo , Zinc , Animales , Modelos Animales de Enfermedad , Masculino , Daño por Reperfusión Miocárdica/sangre , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/fisiopatología , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Factores de Tiempo , Troponina T/sangreRESUMEN
BACKGROUND: Recent studies have shown the potential of PDE-5 inhibition on acute and chronic heart failure. Nevertheless it remained unclear, how far load-reducing properties and direct effects on myocardial contractility are responsible for these observations. In the present study, we investigated the effects of vardenafil on myocardial contractility and vascular function in a dose-response study. METHODS: We performed left ventricular pressure-volume analysis in young adult rats by using a Millar microtip conductance catheter. Pressure-volume loops were recorded before and after intravenous injection of vardenafil (3, 10, 30, 100, 300 µg/kg, n = 6/group). RESULTS: Treatment with vardenafil resulted in a significant (p < 0.05) increase in the load-independent cardiac contractility parameters reaching its maximum at the dose of 100µg/kg (ESPVR: 2.15 ± 0.15 vs. 3.29 ± 0.26 mm Hg/µL; PRSW: 93.28 ± 4.04 vs. 134.90 ± 6.27 mm Hg; peak positive dP/dt/EDV: 38.73 ± 7.97 vs. 53.02 ± 3.74 mm Hg·s-1·µL-1; before versus after 100 µg/kg vardenafil). Results of the in vitro organ-bath experiments showed an augmented vasorelaxation of precontracted aortic rings after vardenafil treatment. CONCLUSION: Our data supports the hypothesis that the usage of vardenafil as "inodilators" could have beneficial effects in heart failure patients.
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Aorta/efectos de los fármacos , Cardiotónicos/farmacología , Imidazoles/farmacología , Contracción Miocárdica/efectos de los fármacos , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Función Ventricular Izquierda/efectos de los fármacos , Animales , Cateterismo Cardíaco , Cardiotónicos/administración & dosificación , Relación Dosis-Respuesta a Droga , Frecuencia Cardíaca/efectos de los fármacos , Imidazoles/administración & dosificación , Técnicas In Vitro , Inyecciones Intravenosas , Masculino , Miografía , Inhibidores de Fosfodiesterasa 5/administración & dosificación , Piperazinas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Volumen Sistólico/efectos de los fármacos , Sulfonas/administración & dosificación , Sulfonas/farmacología , Factores de Tiempo , Triazinas/administración & dosificación , Triazinas/farmacología , Diclorhidrato de Vardenafil , Vasodilatadores/administración & dosificación , Presión Ventricular/efectos de los fármacosRESUMEN
BACKGROUND: The role of the nitric oxide/cGMP/cGMP-dependent protein kinase G pathway in myocardial protection and preconditioning has been the object of intensive investigations. The novel soluble guanylate cyclase activator cinaciguat has been reported to elevate intracellular [cGMP] and activate the nitric oxide/cGMP/cGMP-dependent protein kinase G pathway in vivo. We investigated the effects of cinaciguat on myocardial infarction induced by isoproterenol in rats. METHODS AND RESULTS: Rats were treated orally twice a day for 4 days with vehicle or cinaciguat (10 mg/kg). Isoproterenol (85 mg/kg) was injected subcutaneously 2 days after the first treatment at an interval of 24 hours for 2 days to produce myocardial infarction. After 17 hours, histopathological observations and left ventricular pressure-volume analysis to assess cardiac function with a Millar microtip pressure-volume conductance catheter were performed, and levels of biochemicals of the heart tissues were measured. Gene expression analysis was performed by quantitative real-time polymerase chain reaction. Isolated canine coronary arterial rings exposed to peroxynitrite were investigated for vasomotor function, and immunohistochemistry was performed for cGMP and nitrotyrosine. The present results show that cinaciguat treatment improves histopathological lesions, improves cardiac performance, improves impaired cardiac relaxation, reduces oxidative stress, ameliorates intracellular enzyme release, and decreases cyclooxygenase 2, transforming growth factor-beta, and beta-actin mRNA expression in experimentally induced myocardial infarction in rats. In vitro exposure of coronary arteries to peroxynitrite resulted in an impairment of endothelium-dependent vasorelaxation, increased nitro-oxidative stress, and reduced intracellular cGMP levels, which were all improved by cinaciguat. A cardioprotective effect of postischemic cinaciguat treatment was shown in a canine model of global ischemia/reperfusion. CONCLUSIONS: Pharmacological soluble guanylate cyclase activation could be a novel approach for the prevention and treatment of ischemic heart disease.
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Benzoatos/farmacología , Cardiotónicos/farmacología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/agonistas , Agonistas Adrenérgicos beta/toxicidad , Animales , AMP Cíclico/sangre , GMP Cíclico/sangre , Ciclooxigenasa 2/genética , Perros , Femenino , Expresión Génica/efectos de los fármacos , Guanilato Ciclasa/metabolismo , Pruebas de Función Cardíaca/efectos de los fármacos , Isoproterenol/toxicidad , L-Lactato Deshidrogenasa/sangre , Masculino , Infarto del Miocardio/inducido químicamente , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/inducido químicamente , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa Soluble , Tasa de Supervivencia , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
The muscle Lim protein knock-out (MLP-KO) mouse model is extensively used for studying the pathophysiology of dilated cardiomyopathy. However, explanation is lacking for the observed long survival of the diseased mice which develop until adulthood despite the gene defect, which theoretically predestines them to early death due to heart failure. We hypothesized that adaptive changes of cardiac intracellular calcium (Ca(i)(2+)) handling might explain the phenomenon. In order to study the progression of changes in cardiac function and Ca(i)(2+) cycling, myocardial Ca(i)(2+)-transients recorded by Indo-1 surface fluorometry were assessed with concomitant measurement of hemodynamic performance in isolated Langendorff-perfused hearts of 3- and 9-month old MLP-KO animals. Hearts were challenged with beta-agonist isoproterenol and the sarcoplasmic reticular Ca(2+)-ATPase (SERCA2a) inhibitor cyclopiazonic acid (CPA). Cardiac mRNA content and levels of key Ca(2+) handling proteins were also measured. A decline in lusitropic function was observed in 3-month old, but not in 9-month old MLP-KO mice under unchallenged conditions. beta-adrenergic responses to isoproterenol were similar in all the studied groups. The CPA induced an increase in end-diastolic Ca(i)(2+)-level and a decrease in Ca(2+)-sequestration capacity in 3-month old MLP-KO mice compared to age-matched controls. This unfavorable condition was absent at 9 months of age. SERCA2a expression was lower in 3-month old MLP-KO than in the corresponding controls and in 9-month old MLP-KO hearts. Our results show time-related recovery of hemodynamic function and an age-dependent compensatory upregulation of Ca(i)(2+) handling in hearts of MLP-KO mice, which most likely involve the normalization of the expression of SERCA2a in the affected hearts.
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Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/mortalidad , Corazón/fisiopatología , Hemodinámica , Proteínas Musculares/fisiología , Factores de Edad , Animales , Western Blotting , Índice de Masa Corporal , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Insuficiencia Cardíaca/patología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Indoles/farmacología , Isoproterenol/farmacología , Proteínas con Dominio LIM , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Miocardio/patología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tasa de SupervivenciaRESUMEN
Diabetes mellitus (DM) is associated with characteristic structural and functional changes of the myocardium, termed diabetic cardiomyopathy. As a distinct entity independent of coronary atherosclerosis, diabetic cardiomyopathy is an increasingly recognized cause of heart failure. A detailed understanding of diabetic cardiac dysfunction, using relevant animal models, is required for the effective prevention and treatment of cardiovascular complications in diabetic patients. We investigated and compared cardiac performance in rat models of type 1 DM (streptozotocin induced) and type 2 DM (Zucker diabetic fatty rats) using a pressure-volume (P-V) conductance catheter system. Left ventricular (LV) systolic and diastolic function was evaluated in vivo at different preloads, including the slope of the end-systolic P-V relation (ESPVR) and end-diastolic P-V relationship (EDPVR), preload recruitable stroke work (PRSW), maximal slope of the systolic pressure increment (dP/dt(max)), and its relation to end-diastolic volume (dP/dt(max)-EDV) as well as the time constant of LV relaxation and maximal slope of the diastolic pressure decrement. Type 1 DM was associated with decreased LV systolic pressure, dP/dt(max), slope of ESPVR and dP/dt(max)-EDV, PRSW, ejection fraction, and cardiac and stroke work indexes, indicating marked systolic dysfunction. In type 2 DM rats, systolic indexes were altered only to a lower extent and the increase of LV stiffness was more pronounced, as indicated by the higher slopes of EDPVR. Our data suggest that DM is characterized by decreased systolic performance and delayed relaxation (mainly in type 1 DM), accompanied by increased diastolic stiffness of the heart (more remarkably in type 2 DM). Based on the sophisticated method of P-V analysis, different characteristics of type 1 and type 2 diabetic cardiac dysfunction can be demonstrated.
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Presión Sanguínea/fisiología , Volumen Sanguíneo/fisiología , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Función Ventricular Izquierda/fisiología , Animales , Glucemia/metabolismo , Peso Corporal/fisiología , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 2/genética , Glucosuria/metabolismo , Masculino , Contracción Miocárdica/fisiología , Ratas , Ratas Sprague-Dawley , Ratas ZuckerRESUMEN
OBJECTIVES: Vascular grafts are often stored in cold physiological saline/heparinized blood preservation solution. Until now, only in vitro studies investigated the effect of the aforementioned preservation solutions on endothelial function. The main goal of our study was to compare the storage effect of physiological saline and heparinized blood after short-time cold storage and warm reperfusion in a rat model of aortic transplantation. METHODS: Aortic abdominal transplantations (n = 6-8/group) were performed in Lewis rats. The donor aortic arches were placed in cold physiological saline and heparinized blood solutions and stored for 2 h. After the 2 h ischaemia, the aortic arches were transplanted into the abdominal aorta of the recipient. Two, 24 h or 1 week after transplantation, implanted grafts were harvested. Endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) vasorelaxation were investigated in organ bath experiments. DNA strand breaks were assessed by transferase-mediated dUTP nick-end labelling-method and mRNA expression by quantitative real-time polymerase chain reaction. In addition, the expression of CD-31 was also investigated by immunochemistry. RESULTS: Severely impaired endothelial function and integrity of grafts were shown after 2 and 24 h reperfusion in both groups (maximal vasorelaxation control: 94 ± 1%, heparinized blood: 27 ± 4 and 17 ± 3%, saline 34 ± 5% and 28 ± 5%; CD-31 positive area control: 96 ± 1% blood: 38 ± 8% and 41 ± 6%, saline: 35 ± 12% and 41 ± 7%, respectively P < 0.05). After 1 week, endothelial function and integrity were partially recovered (maximal vasorelaxation: heparinized blood: 46 ± 4%, saline: 46 ± 2%, CD-31 positive area blood: 35 ± 4%; saline: 56 ± 5%, P < 0.05). In addition, mRNA levels of Bax, Bcl-2 and caspase-3 were significantly altered and DNA stand breaks were observed. CONCLUSIONS: Storage with the generally used physiological saline and heparinized blood solutions is unable to protect the endothelium against cold ischaemia and warm reperfusion injury. A similar weak preservation effect was observed.
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Aorta Abdominal , Rechazo de Injerto/etiología , Soluciones Preservantes de Órganos/efectos adversos , Soluciones Preservantes de Órganos/farmacología , Preservación de Órganos/métodos , Injerto Vascular/métodos , Análisis de Varianza , Animales , Criopreservación/métodos , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Heparina/efectos adversos , Heparina/farmacología , Masculino , Preservación de Órganos/efectos adversos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , ARN Mensajero/análisis , Distribución Aleatoria , Ratas , Ratas Endogámicas Lew , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Cloruro de Sodio/farmacología , Injerto Vascular/efectos adversos , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND: Although, ischemia/reperfusion induced vascular dysfunction has been widely described, no comparative study of in vivo- and in vitro-models exist. In this study, we provide a direct comparison between models (A) ischemic storage and in-vitro reoxygenation (B) ischemic storage and in vitro reperfusion (C) ischemic storage and in-vivo reperfusion. METHODS AND RESULTS: Aortic arches from rats were stored for 2 hours in saline. Arches were then (A) in vitro reoxygenated (B) in vitro incubated in hypochlorite for 30 minutes (C) in vivo reperfused after heterotransplantation (2, 24 hours and 7 days reperfusion). Endothelium-dependent and independent vasorelaxations were assessed in organ bath. DNA strand breaks were assessed by TUNEL-method, mRNA expressions (caspase-3, bax, bcl-2, eNOS) by quantitative real-time PCR, proteins by Western blot analysis and the expression of CD-31 by immunochemistry. Endothelium-dependent maximal relaxation was drastically reduced in the in-vivo models compared to ischemic storage and in-vitro reperfusion group, and no difference showed between ischemic storage and control group. CD31-staining showed significantly lower endothelium surface ratio in-vivo, which correlated with TUNEL-positive ratio. Increased mRNA and protein levels of pro- and anti-apoptotic gens indicated a significantly higher damage in the in-vivo models. CONCLUSION: Even short-period of ischemia induces severe endothelial damage (in-vivo reperfusion model). In-vitro models of ischemia-reperfusion injury can be limitedly suited for reliable investigations. Time course of endothelial stunning is also described.
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Aorta/metabolismo , Endotelio Vascular/metabolismo , Daño por Reperfusión/genética , Animales , Aorta/efectos de los fármacos , Aorta/patología , Aorta/trasplante , Apoptosis/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Regulación de la Expresión Génica , Ácido Hipocloroso/farmacología , Etiquetado Corte-Fin in Situ , Masculino , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Oxígeno/farmacología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transducción de Señal , Técnicas de Cultivo de Tejidos , Trasplante Heterólogo , Vasodilatación/efectos de los fármacosRESUMEN
The pathophysiology of ischemic myocardial injury involves cellular events, reactive oxygen species, and an inflammatory reaction cascade. The zinc complex of acetylsalicylic acid (Zn(ASA)2) has been found to possess higher anti-inflammatory and lower ulcerogenic activities than acetylsalicylic acid (ASA). Herein, we studied the effects of both ASA and Zn(ASA)2 against acute myocardial ischemia. Rats were pretreated with ASA (75 mg/kg) or Zn(ASA)2 (100 mg/kg) orally for five consecutive days. Isoproterenol (85 mg/kg, subcutaneously [s.c.]) was applied to produce myocardial infarction. After 17-22 h, animals were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally [i.p.]) and both electrical and mechanical parameters of cardiac function were evaluated in vivo. Myocardial histological and gene expression analyses were performed. In isoproterenol-treated rats, Zn(ASA)2 treatment normalized significantly impaired left-ventricular contractility index (Emax 2.6 ± 0.7 mmHg/µL vs. 4.6 ± 0.5 mmHg/µL, P < 0.05), increased stroke volume (30 ± 3 µL vs. 50 ± 6 µL, P < 0.05), decreased systemic vascular resistance (7.2 ± 0.7 mmHg/min/mL vs. 4.2 ± 0.5 mmHg/min/mL, P < 0.05) and reduced inflammatory infiltrate into the myocardial tissues. ECG revealed a restoration of elevated ST-segment (0.21 ± 0.03 mV vs. 0.09 ± 0.02 mV, P < 0.05) and prolonged QT-interval (79.2 ± 3.2 ms vs. 69.5 ± 2.5 ms, P < 0.05) by Zn(ASA)2. ASA treatment did not result in an improvement of these parameters. Additionally, Zn(ASA)2 significantly increased the mRNA-expression of superoxide dismutase 1 (+73 ± 15%), glutathione peroxidase 4 (+44 ± 12%), and transforming growth factor (TGF)-ß1 (+102 ± 22%). In conclusion, our data demonstrate that oral administration of zinc and ASA in the form of bis(aspirinato)zinc(II) complex is superior to ASA in preventing electrical, mechanical, and histological changes after acute myocardial ischemia. The induction of antioxidant enzymes and the anti-inflammatory cytokine TGF-ß1 may play a pivotal role in the mechanism of action of Zn(ASA)2.
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Aspirina/análogos & derivados , Complejos de Coordinación/administración & dosificación , Isquemia Miocárdica/prevención & control , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Aspirina/administración & dosificación , Electrocardiografía , Regulación de la Expresión Génica/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Isoproterenol/toxicidad , Masculino , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/inducido químicamente , Infarto del Miocardio/complicaciones , Infarto del Miocardio/patología , Isquemia Miocárdica/etiología , Isquemia Miocárdica/fisiopatología , Ratas , Ratas Sprague-Dawley , Troponina T/sangre , Zinc/administración & dosificaciónRESUMEN
BACKGROUND: The nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is an important key mechanism to protect the heart from ischemia/reperfusion injury. However, this pathway is disrupted in several cardiovascular diseases as a result of decreased NO bioavailability and increased NO-insensitive forms of sGC. Cinaciguat preferentially activates these NO-insensitive, oxidized forms of sGC. METHODS: We assessed the hypothesis that targeting NO-unresponsive sGC would protect the graft against ischemia/reperfusion injury in a rat heart transplantation model. Before explantation, donor Lewis rats received methylcellulose (1%) vehicle or cinaciguat 10 mg/kg. The hearts were excised, stored in cold preservation solution, and heterotopically transplanted. We evaluated in vivo left ventricular function of the graft. RESULTS: After transplantation, decreased left ventricular systolic pressure (77 ± 3 mm Hg vs 123 ± 13 mm Hg, p < 0.05), dP/dt(max) (1,703 ± 162 mm Hg vs 3,350 ± 444 mm Hg, p < 0.05), and dP/dt(min) (995 ± 110 mm Hg vs 1,925 ± 332 mm Hg, p < 0.05) were significantly increased by cinaciguat. Coronary blood flow was significantly higher in the cinaciguat group compared with the control group. Additionally, cinaciguat increased adenosine triphosphate levels (1.9 ± 0.4 µmol/g vs 6.6 ± 0.8 µmol/g, p < 0.05) and improved energy charge potential. After transplantation, increased c-jun messenger RNA expression was downregulated, whereas superoxide dismutase-1 and cytochrome-c oxidase mRNA levels were upregulated by cinaciguat. Cinaciguat also significantly decreased myocardial DNA strand breaks induced by ischemia/reperfusion during transplantation and reduced death of cardiomyocytes in a cellular model of oxidative stress. CONCLUSIONS: By interacting with NO-unresponsive sGC, cinaciguat enhances the protective effects of the NO/cGMP pathway at different steps of signal transduction after global myocardial ischemia/reperfusion. Its clinical use as pre-conditioning agent could be a novel approach in cardiac surgery.
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Benzoatos/uso terapéutico , Guanilato Ciclasa/fisiología , Trasplante de Corazón/efectos adversos , Daño por Reperfusión Miocárdica/prevención & control , Receptores Citoplasmáticos y Nucleares/fisiología , Acondicionamiento Pretrasplante , Animales , Activación Enzimática , Masculino , Daño por Reperfusión Miocárdica/etiología , Ratas , Ratas Endogámicas Lew , Guanilil Ciclasa Soluble , Función Ventricular IzquierdaRESUMEN
Increasing evidence suggests that both types of diabetes mellitus (DM) lead to cardiac structural and functional changes. In this study we investigated and compared functional characteristics and underlying subcellular pathological features in rat models of type-1 and type-2 diabetic cardiomyopathy. Type-1 DM was induced by streptozotocin. For type-2 DM, Zucker Diabetic Fatty (ZDF) rats were used. Left ventricular pressure-volume analysis was performed to assess cardiac function. Myocardial nitrotyrosine immunohistochemistry, TUNEL assay, hematoxylin-eosin, and Masson's trichrome staining were performed. mRNA and protein expression were quantified by qRT-PCR and Western blot. Marked systolic dysfunction in type-1 DM was associated with severe nitrooxidative stress, apoptosis, and fibrosis. These pathological features were less pronounced or absent, while cardiomyocyte hypertrophy was comparable in type-2 DM, which was associated with unaltered systolic function and increased diastolic stiffness. mRNA-expression of hypertrophy markers c-fos, c-jun, and ß-MHC, as well as pro-apoptotic caspase-12, was elevated in type-1, while it remained unaltered or only slightly increased in type-2 DM. Expression of the profibrotic TGF-ß 1 was upregulated in type-1 and showed a decrease in type-2 DM. We compared type-1 and type-2 diabetic cardiomyopathy in standard rat models and described an altered pattern of key pathophysiological features in the diabetic heart and corresponding functional consequences.
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Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Cardiopatías/complicaciones , Miocardio/patología , Animales , Apoptosis , Complicaciones de la Diabetes/fisiopatología , Diabetes Mellitus Experimental/fisiopatología , Fibrosis , Regulación de la Expresión Génica , Corazón/fisiopatología , Hemodinámica , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Estrés Oxidativo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Zucker , Factor de Crecimiento Transformador beta1/metabolismo , Tirosina/análogos & derivados , Tirosina/química , Función Ventricular IzquierdaRESUMEN
UNLABELLED: In recent years, there has been a growing interest in molecular imaging markers of tumor-induced angiogenesis. Several radiolabeled RGD (arginine, glycine, aspartate) peptides have been developed for PET imaging of αvß3 integrins in the tumor vasculature, but there are only limited data on how angiogenesis inhibitors affect the tumor uptake of these peptides. METHODS: Changes in (68)Ga-NODAGA-c(RGDfK) peptide uptake were measured using PET during bevacizumab therapy of 2 αvß3-negative squamous cell carcinoma cell lines (A-431 and FaDu) that induce αvß3-positive neovasculature when transplanted into nude mice. Tumor uptake of (68)Ga-NODAGA-c(RGDfK) was correlated to microvascular density, vascular morphology, and permeability as well as αvß3 integrin expression. RESULTS: Bevacizumab significantly inhibited growth of A-431 tumors and caused a significant reduction in microvascular density and αvß3 integrin expression within 7 d after start of therapy. Bevacizumab also caused a normalization of blood vessel morphology and decreased tumor necrosis. However, (68)Ga-NODAGA-c(RGDfK) uptake was significantly increased at day 7 of therapy and did not decrease until after 3 wk of treatment. In Fadu xenografts, bevacizumab therapy caused only a minor inhibition of tumor growth and minor changes in (68)Ga-NODAGA-c(RGDfK) uptake. CONCLUSION: Uptake of radiolabeled RGD peptides is not necessarily decreased by effective antiangiogenic therapy. Early in the course of therapy a decrease in the expression of αvß3 integrins may not be reflected by a decrease in the uptake of RGD peptides.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/tratamiento farmacológico , Integrina alfaVbeta3/metabolismo , Neovascularización Patológica , Tomografía de Emisión de Positrones , Acetatos , Algoritmos , Inhibidores de la Angiogénesis/química , Animales , Bevacizumab , Línea Celular Tumoral , Femenino , Radioisótopos de Galio/química , Compuestos Heterocíclicos con 1 Anillo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microcirculación , Necrosis , Trasplante de Neoplasias , Oligopéptidos/química , Péptidos/química , Permeabilidad , Factores de TiempoRESUMEN
Reactive oxygen species, such as hypochlorite induce oxidative stress, which impairs nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling and leads to vascular dysfunction. It has been proposed, that elevated cGMP-levels may contribute to an effective cytoprotection against oxidative stress. We investigated the effects of vardenafil, a selective inhibitor of the cGMP-degrading phosphodiesterase-5 enzyme on vascular dysfunction induced by hypochlorite. In organ bath experiments for isometric tension, we investigated the endothelium-dependent and endothelium-independent vasorelaxation of isolated rat aortic rings using cumulative concentrations of acetylcholine and sodium nitroprusside (SNP). Vascular dysfunction was induced by exposing rings to hypochlorite (100-400 µM). In the treatment groups, rats were pretreated with vardenafil (30 and 300 µg/kg i.v.). Immunohistochemical analysis was performed for the oxidative stress markers nitrotyrosine, poly(ADP-ribose) and for apoptosis inducing factor (AIF). Exposure to hypochlorite resulted in a marked impairment of acetylcholine-induced endothelium-dependent vasorelaxation of aortic rings. Pretreatment with vardenafil led to improved endothelial function as reflected by the higher maximal vasorelaxation (Rmax) to acetylcholine. Regarding endothelium-independent vasorelaxation, hypochlorite exposure led to a left-shift of SNP concentration-response curves in the vardenafil groups without any alterations of the Rmax. In the hypochlorite groups immunohistochemical analysis showed enhanced poly(ADP-ribose)-formation and nuclear translocation of AIF, which were prevented by vardenafil-pretreatment. Our results support the view that cytoprotective effects of PDE-5-inhibitors on the endothelium may underlie the improved endothelial function, however, a slight sensitisation of vascular smooth muscle to NO was also confirmed. PDE-5-inhibition may represent a potential therapy approach for treating vascular dysfunction induced by oxidative stress.
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Endotelio Vascular/efectos de los fármacos , Imidazoles/farmacología , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/farmacología , Acetilcolina/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Endotelio Vascular/fisiopatología , Ácido Hipocloroso , Técnicas In Vitro , Masculino , Nitroprusiato/farmacología , Oxidantes , Estrés Oxidativo/efectos de los fármacos , Fenilefrina/farmacología , Ratas , Ratas Sprague-Dawley , Sulfonas/farmacología , Triazinas/farmacología , Diclorhidrato de Vardenafil , Vasoconstrictores/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Oxidative stress interferes with nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) signalling pathway through reduction of endogenous NO and formation of the strong intermediate oxidant peroxynitrite and leads to vascular dysfunction. We evaluated the effects of oral treatment with NO- and heme-independent sGC activator cinaciguat on peroxynitrite-induced vascular dysfunction in rat aorta. Sprague-Dawley rats were treated orally 2 times at an interval of 17 hours with vehicle or with cinaciguat (10 mg/kg). One hour after the last treatment, the animals were anesthetized, the thoracic aorta was removed, and the aortic segment preparations were incubated with and without the reactive oxidant peroxynitrite (200 µmol/L, 30 minutes). Endothelium-dependent (acetylcholine), -independent (sodium nitroprusside) vasorelaxations were investigated, and histopathological examination was performed. Incubation of aortic rings with peroxynitrite significantly attenuated the maximal endothelium-dependent relaxation (R (max)) to acetylcholine (peroxynitrite, 44.5% ± 5.9% vs control, 93.2% ± 2.0%, P < .05) and decreased pD(2) values (-logEC(50), EC(50) being the concentration of acetylcholine that elicited 50% of the maximal response) for the concentration-response curves as compared to control segments. Treatment of rats with cinaciguat significantly improved the decreased acetylcholine-induced vasorelaxation after exposure of aortic rings to peroxynitrite (cinaciguat + peroxynitrite, 67.1% ± 3.5% vs peroxynitrite, 44.5% ± 5.9%, P < .05). Incubation of aortic segments with peroxynitrite caused a significant shift of the sodium nitroprusside concentration-response curves to the right without any alterations in the R (max). Moreover, exposure of aortic rings to peroxynitrite resulted in increased nitro-oxidative stress and DNA breakage which were improved by cinaciguat. Treatment of rats with cinaciguat significantly increased intracellular cGMP levels in the aortic wall. Our results show under conditions of nitro-oxidative stress when signalling in the NO/sGC/cGMP pathway is impaired, acute activation of sGC by cinaciguat might be advantageous in the treatment of endothelial dysfunction in cardiovascular disease.
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Aorta Torácica/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Guanilato Ciclasa/fisiología , Hemo/fisiología , Óxido Nítrico/fisiología , Ácido Peroxinitroso/toxicidad , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Aorta Torácica/fisiología , Benzoatos/farmacología , GMP Cíclico/análisis , GMP Cíclico/fisiología , Roturas del ADN , Endotelio Vascular/fisiología , Técnicas In Vitro , Masculino , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Guanilil Ciclasa Soluble , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND: Many donor organs come from youths involved in alcohol-related accidental death. The use of cardiac allografts for transplantation from donors after acute poisoning is still under discussion while acute ethanol intoxication is associated with myocardial functional and morphological changes. The aims of this work were 1) to evaluate in rats the time-course cardiac effects of acute ethanol-exposure and 2) to explore how its abuse by donors might affect recipients in cardiac pump function after transplantation. METHODS: Rats received saline or ethanol (3.45 g/kg, ip). We evaluated both the mechanical and electrical aspects of cardiac function 1 h, 6 h or 24 h after injection. Plasma cardiac troponin-T and glucose-levels were measured and histological examination of the myocardium was performed. In addition, heart transplantation was performed, in which donors received ethanol 6 h or 24 h prior to explantation. Graft function was measured 1 h or 24 h after transplantation. Myocardial TBARS-concentration was measured; mRNA and protein expression was assessed by quantitative real-time PCR and Western blot, respectively. RESULTS: Ethanol administration resulted in decreased load-dependent (-34 ± 9%) and load-independent (-33 ± 12%) contractility parameters, LV end-diastolic pressure and elevated blood glucose levels at 1 h, which were reversed to the level of controls after 6 h and 24 h. In contrast to systolic dysfunction, active relaxation and passive stiffness are slowly recovered or sustained during 24 h. Moreover, troponin-T-levels were increased at 1 h, 6 h and 24 h after ethanol injection. ST-segment elevation (+47 ± 10%), elongated QT-interval (+38 ± 4%), enlarged cardiomyocyte, DNA-strand breaks, increased both mRNA and protein levels of superoxide dismutase-1, glutathione peroxydase-4, cytochrome-c-oxidase and metalloproteinase-9 were observed 24 h following ethanol-exposure. After heart transplantation, decreased myocardial contractility and relaxation, oxidative stress and altered protein expression were observed. CONCLUSIONS: These results demonstrate acute alcohol abuse increases the susceptibility of donor hearts to ischemia/reperfusion in a rat heart transplant model even though the global contractile function recovers 6 h after ethanol-administration.
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Etanol/efectos adversos , Trasplante de Corazón/efectos adversos , Corazón/efectos de los fármacos , Daño por Reperfusión Miocárdica/etiología , Miocardio/metabolismo , Daño por Reperfusión/etiología , Animales , Etanol/administración & dosificación , Corazón/fisiopatología , Hemodinámica/efectos de los fármacos , Masculino , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Ratas , Ratas Endogámicas Lew , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Troponina I/metabolismoRESUMEN
Reactive oxygen species, such as peroxynitrite, induce oxidative stress and DNA injury leading to endothelial dysfunction. It has been proposed, that elevated intracellular cyclic GMP (cGMP)-levels may contribute to an effective cytoprotection against nitro-oxidative stress. We investigated the dose-dependent effects of vardenafil, an inhibitor of phosphodiesterase-5, on endothelial dysfunction induced by peroxynitrite. In organ bath experiments, we investigated the endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside, SNP) vasorelaxation of isolated aortic rings of rats. Endothelial dysfunction was induced by peroxynitrite. In the treatment groups, rats received low doses (0.01-5 microg/kg) or high doses (5-300 microg/kg) of vardenafil. DNA strand breaks were assessed by the TUNEL method. Immunohistochemical analysis was performed for cGMP and nitrotyrosine. Exposure to peroxynitrite resulted in an impairment of endothelium-dependent vasorelaxation of aortic rings. Pre-treatment with lower doses of vardenafil led to an improvement of endothelial function as reflected by the higher maximal vasorelaxation (R(max)) to acetylcholine. Interestingly, at higher doses, R(max) to acetylcholine was attenuated leading to U-shaped dose-response curves. The endothelium-independent vasorelaxation to SNP under peroxynitrite stress showed a significant left-shift of the SNP concentration-response curves in the vardenafil groups without any alterations of the R(max). Vardenafil-pre-treatment significantly reduced DNA-breakage, reduced nitrosative stress, and increased cGMP score in the aortic wall. Our working hypothesis is that improvement of endothelial function could be mainly due to the cytoprotection of endothelium by vardenafil. This work supports the view that acute PDE5-inhibition might be advantageous in the treatment of endothelial dysfunction induced by disturbed NO-cGMP pathway due to nitro-oxidative stress.
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Aorta Torácica/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Imidazoles/farmacología , Ácido Peroxinitroso/metabolismo , Inhibidores de Fosfodiesterasa 5 , Inhibidores de Fosfodiesterasa/farmacología , Piperazinas/farmacología , Vasodilatadores/farmacología , Acetilcolina/farmacología , Animales , Aorta Torácica/fisiología , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/fisiología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Técnicas In Vitro , Masculino , Nitroprusiato/farmacología , Estrés Oxidativo/efectos de los fármacos , Ácido Peroxinitroso/farmacología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Sulfonas/farmacología , Triazinas/farmacología , Diclorhidrato de Vardenafil , Vasodilatación/efectos de los fármacosRESUMEN
Recently, the infarct reducing and cardioprotective effects of phosphodiesterase-5-inhibitors were described. In this study, we investigated these effects on ischemia/reperfusion injury in a rat model of heart transplantation. Three groups were assigned for our study: a vardenafil preconditioning group, an ischemic control, and a nonischemic control. Hemodynamic parameters were significantly increased in the vardenafil group (Pmax: 82+/-4 vs. 110+/-12 vs. 127+/-13 mm Hg; dP/dtmax: 1740+/-116 vs. 3197+/-599 vs. 4397+/-602 mm Hg/sec; ischemic control vs. vardenafil vs. nonischemic control; P<0.05 vs. ischemic control). Furthermore, we recorded increased ATP levels and significantly less apoptosis in the treatment group after terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (apoptosis index: 27.23%+/-1.54% vs. 16.77%+/-1.42% vs. 18.86%+/-1.07%; ischemic control vs. vardenafil vs. nonischemic control; P<0.05 vs. ischemic control). Our current results support the concept that the cGMP-PKG-pathway plays an important role in ischemia/reperfusion injury. We could show that up-regulating this pathway has a preconditioning-like effect and can effectively reduce ischemia/reperfusion injury.